Na+ - dependent nucleoside transporters mediate the salvage of purine and pyrimidine nucleosides in mammalian cells. These transporters are also important in the absorption, disposition, and targeting of therapeutic nucleoside analogs. Five subtypes of Na+ - dependent nucleoside transporters have been characterized. Subtype N1 is purine selective and subtype N2 is pyrimidine selective. Both of these structures have been predicted to possess 14 putative transmembrane domains based on cDNA and hydropathy analysis. Using chimeric rat N1/N2 constructs, it was determined that transmembrane domains 8 and 9 are the major site for substrate binding. Mutational analysis of TM8 demonstrated that Ser318 was primarily responsible for the pyrimidine specificity of N2, and it was proposed to act as a gating residue in an aqueous channel. We are using both homology modeling and ab initio structure generation to generate models for these structures in order to: 1) test these current structural hypotheses and 2) to construct experiments which will further elucidate structural and mechanistic details of these transporters.